Fructose-Enhanced Development and Growth of the N2-Fixing Cyanobiont Anabaena azollae Anat Rozen, Mordechay Schönfeld, and Elisha Tel-Or Department of Agricultural Botany, Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel

Z. Naturforsch. 43c, 408-412 (1988); received February 1, 1988

Nitrogen Fixation, Symbiotic Cyanobacteria, Photosynthesis, Respiration, Heterotrophic Growth Fructose supported the heterotrophic growth of the cyanobiont Anabaena azollae, isolated

from the water fern Azolla filiculoides, and also enhanced its growth in the light by 2-3- fo ld . Fructose was taken up at a high rate in the light and in the dark, in an energy-dependent reaction. The photosynthetic and respiratory activities of the fructose grown cells were modified: 0 2 evolu-tion in vivo was decreased by 40%, while PS I activity and dark respiration were 2 -3- fo ld higher than in autotrophically grown cells. These changes were accompanied by 2-3-fo ld increase in heterocyst differentiation and by a 4-fold stimulation of nitrogenase activity.

Introduction

The symbiotic relationship between the water fern Azolla and the N2-fixing cyanobiont Anabaena azol-lae has been extensively studied, and it is generally accepted that the cyanobiont is supported by photo-synthates provided by the host [1]. Extracts of the host fern Azolla were found to contain high concen-trations of sucrose, fructose and glucose, which are assumed to be available to the cyanobiont in the leaf cavity [2, 3], Fructose was previously observed to support growth in Anabaena azollae isolated from Azolla caroliniana [4] and Azolla filiculoides [5], The ability to take up and metabolize exogenous sugars, and utilize them as major carbon sources for energy supply and growth, is therefore considered a prerequi-site for the symbiotic relationship of Anabaena azollae with its host Azolla.

Photosynthates supplied by the host might be expected to modify the energy metabolism of the cyanobiont, increasing respiratory activity and de-creasing photosynthesis and altogether inducing a heterotrophic-growth pattern. Currently there is very little known on the effects of photosynthates provided by Azolla on the respiratory and photo-synthetic activities of the cyanobiont. There is also practically no information on the photosynthetic ac-tivity of cyanobacteria under conditions of sugar sup-

Abbreviations: CCCP, (carbonylcyanide m-chlorophenyl-hydrazone); DCMU, [3-(3,4-dichlorophenyl)-l,l-dimeth-ylurea].

Reprint requests to Prof. Elisha Tel-Or.

Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 0341 - 0382/88/0500 - 0408 S 01.30/0

ported growth, either in the light or in the dark, and only limited information on respiratory activity under such conditions [6, 7],

Modifications induced by exogenous sugars in the metabolism of the cyanobiont, are also expected to include enhancement of nitrogen fixation, which is the main contribution of the cyanobiont to the sym-biosis. Enhancement of nitrogenase activity by fruc-tose and sucrose was indeed reported for Anabaena azollae, isolated from Azolla caroliniana, Azolla filiculoides and Azolla pinnata [8-10]. Growth of Anabaena variabilis in the presence of fructose, was found to involve accumulation of glycogen. It was suggested that glycogen accumulation is necessary to stabilize nitrogenase activity [11, 12]. An increased nitrogenase activity accompanied by glycogen ac-cumulation was also found in fructose-grown Anabaena azollae [5].

In experiments described in this communication, we tested the effects of fructose on growth and devel-opment of A. azollae in the dark and in the light, and analyzed the responses to fructose of N2-fixation, photosynthesis and respiration in these cells. Growth of Anabaena azollae in the presence of fructose in-duced major modifications in its photosynthetic and respiratory activities, adapting it to heterotrophic growth and to its role as the nitrogen fixing partner in the symbiosis.

Materials and Methods

Organism and cultures

Anabaena azollae was isolated in our laboratory from the water fern Azolla filiculoides [13]. The cul-

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A. Rozen et al. • Fructose-Supported Development of Anabaena azollae 409

ture was routinely grown as described by Tel-Or and Sandovsky [8]. Cells from two week old cultures were transferred to 250 ml Erlenmeyer flasks con-taining BG-11 medium [14], and were grown in the dark or in the light (1 = 5 watt • irT2), with or without added sugar, for one to two weeks.

Analytical procedures and assays

Cells of Anabaena azollae were collected every 24 h for analysis. Representative results from 4—6 experimental series are shown, each result being the average of duplicate or triplicate determinations. Nitrogenase activity, measured by acetylene reduc-tion, cell dry weight and glycogen content was deter-mined as described by Rozen et al. [5]. Sugar content of the medium was determined by the phenol sulfuric acid procedure [15]. Chlorophyll was determined according to Mackinney [16].

Fructose incorporation was studied with [U-14C]fructose. Cells were incubated at room tempera-ture for 15 min in BG-11 medium containing 8 mM fructose with 0.06 pCi [U-14C]fructose per sample, on an illuminated shaker (1 = 5 W-m~2). The cells were collected and washed on a milipore filter (0.45 pm), suspended in scintillation liquid, and analyzed with a Beckman LS 7800-counter. Photo-synthesis and respiration activities in vivo were determined by changes in oxygen concentration as measured by a Clark-type 0 2 electrode (Y.S.I. 5331). Oxygen uptake in dark and evolution in light (1 = 2300 pE • m - 2 • sec - 1) were calculated from the slope of the curve during steady state. PS I activity was determined with protoplasts prepared by lyzo-zyme treatment [17]. The reaction mixture contained 5 mM sodium ascorbate, 50 pM 2,6, dichloro-phenolindophenol, 50 pM methyl viologen, 1 mM NaN3, 10 pM DCMU in 25 mM hepes pH 7.5, and protoplasts containing 20 pg Chi a -ml - 1 . Reaction mixture for assays of PS II activity contained 25 mM Hepes buffer pH 7.5, 1 mM potassium ferricyanide, and protoplasts containing 20 pg Chi a -ml - 1 .

Results and Discussion

Fructose was previously shown to enhance the growth of A. azollae in the light. Cell growth rate in batch cultures was 2—3-fold higher in the presence of 8 mM of fructose, compared to autotrophically grown cells [5]. As shown in Fig. 1, the growth pattern of the cells in the light was similar in the absence and

EFFECT OF 8 mM FRUCTOSE ON THE GROWTH Of

as

0.6

E c o m m 5 0 . 4

ci o

0.2

0

Fig. 1. Effect of 8 mM fructose on the growth of Anabaena azollae. Cells were grown as described in Materials and Methods. O—O, light; 3 — 3 , light +5 (ím DCMU; • — • , dark.

the presence of 5 pM DCMU. In addition, fructose-supported growth in the dark exhibited an initial lag, and the maximal cell density achieved was about half of that obtained in the light. These results seem to suggest that maximal growth in the presence of fruc-tose depended on PS I activity, but was largely inde-pendent of PS II.

Nitrogenase activity of autotrophic cultures, as measured by acetylene reduction, which was usually about 100 pmoles ethylene-g dw _ 1 -h _ 1 , increased 2—4-fold by the presence of fructose in the growth medium. As shown in Fig. 2, nitrogenase activity in fructose-grown cells in the light was largely unaf-fected by DCMU, suggesting that the contribution of PS II to nitrogen fixation was insignificant under these conditions. The maximal activity in the dark was about half of that in the light, suggesting that respiration could only partially support the energetic demands of heterotrophic nitrogen fixation, and that the operation of PS I was essential for optimal ac-tivity.

Maximal nitrogenase activity was obtained be-tween the second and fourth days of growth in the light (Fig. 2), when culture density reached about half of its maximum (0.2—0.4 mg cell dry weight/ml). The increase of nitrogenase activity coincided with a rise in frequency of heterocysts which was highest in the culture grown with 8 mM fructose. Heterocyst frequencies of 5%, 16%, 18%, and 14% were ob-

Anoboena azolloe

410 A. Rozen et al. • Fructose-Supported Development of Anabaena azollae 410

EFFECT OF 8 mM FRUCTOSE ON C?H, REDUCTION ACTIVITY BY Anabaena azollae

Fig. 2. Effect of 8 mM fructose on nitrogenase activity of Anabaena azollae. Activity of acetylene reduction was as-sayed as described in Materials and Methods. O—O, light; C—€), light +5 pM DCMU; • — d a r k ; • — • , light without fructose.

tained for cultures grown in the light with 0, 2.5, 8, and 16 mM fructose respectively. Acetylene reduc-tion activity in the dark exhibited an initial lag and reached its maximum between the fourth and sixth day. The pattern of changes in frequency of hetero-cysts in the dark, corresponded to the pattern of changes in nitrogenase activity. The maximal hetero-cyst frequency in the dark was 14%. A close correla-tion between growth and nitrogenase activity in the dark (Fig. 1 and 2) could suggest that nitrogen fixa-tion is the rate limiting factor for heterotrophic growth.

Fructose-grown A. azollae cells were previously shown to accumulate glycogen during growth in the light [5], and a similar result was obtained in the present study for fructose-grown cells in the dark. The cell glycogen content increased sharply, reach-ing about 20% of the cell dry weight by the second or the third day of growth, and then decreased (data not shown). The apparent correlation between the in-creases in glycogen content and induction of nitro-genase activity agrees with previous reports of such correlation in fructose grown Anabaena variabilis which might indicate stabilization of nitrogenase by

glycogen [11, 12], It was suggested that the high gly-cogen content could support high respiration rates, which would keep oxygen at a low concentration, and thus stabilize nitrogenase activity.

The limited effect of DCMU on fructose-sup-ported growth and on fructose-enhanced nitrogenase activity, raised the question whether growth in the presence of fructose involved modifications of the photosynthetic apparatus resembling those occurring during the process of heterocyst differentiation. En-hanced PS I activity in heterocysts, involving an in-crease in number of PS I reaction-centers per cell is accompanied by a loss of PS II activity [18]. This possibility was tested by measurements of partial photosynthetic reactions in hypotonically broken protoplasts, isolated from either fructose-grown cells or autotrophically grown cells. PS I activity was measured by the light-driven electron transport from reduced dichlorophenolindophenol to methyl violo-gen. The specific activity of preparations from auto-trophically grown cells was 200 peq-mg C h l - I ' h - 1 , while the activity of preparations from cells grown in the light in the presence of fructose was 660 peq • mg Chl _ 1 -h _ 1 . PS II activity was apparently impaired during the isolation process, and no activity of elec-tron transport from water to ferricyanide could be detected in preparations from either control or fruc-tose grown cells. These results thus indicate that fructose induced a major increase in PS I activity but provided no information regarding the effect of fruc-tose on PS II. An indication for fructose-induced re-duction in PS II activity was found in the fact that oxygen evolution by A. azollae cells, grown for four days in the presence of fructose, was about 40% low-er than that of cells grown autotrophically (Table I).

Table I. Photosynthetic 0 2 evolution and respiratory 0 2 consumption by A. azollae.

Cell growth conditions 0 2 uptake 0 2 evolution (dark) (light)

light grown in BG-11 medium 38 155 light grown with 8 mM fructose 50 92 dark grown with 8 mM fructose 90 92

Cells were grown for four days under the specified growth conditions. Respiratory activity was measured in the dark in the absence of exogenous sugar. Reaction rates are in pmoles 0 2 - m g C h r ' - h r - 1 . Assays were conducted with cells, containing 30—50 pg Chl a, suspended in fresh BG-11 medium.

A. Rozen et al. • Fructose-Supported Development of Anabaena azollae 411

These results correlate well with the insensitivity to DCMU of cell growth (Fig. 1) and nitrogenase activity (Fig. 2) in the presence of fructose. The 3-fold higher PS I activity may help to provide the high ATP levels required for fructose uptake and the en-hanced nitrogenase activity. This increase in PS I activity induced by fructose, seems to resemble the enrichment in PS I reaction centers which takes place during differentiation of vegetative cells to hetero-cysts [18]. PS II activity in the latter is lost during differentiation. Preliminary studies on the effect of fructose on the ultrastructure of A. azollae cells have indicated a different arrangement and reduced number of thylakoids in the fructose grown cells (results not shown).

The 2—3-fold increase in growth rate in the light, induced by fructose [5], was not accompanied by a comparable increase in respiration rate, as might have been expected. The respiration of cells grown in the light in the presence of fructose was only 32% higher than that of autotrophically grown cells (Ta-ble I). It might thus be concluded that fructose en-hanced growth in the light, not by serving as a sub-strate for respiration, but rather by enhancing PS I activity. In the dark, on the other hand, fructose was essential as a substrate, and the oxygen consumption rate was indeed two and a half times higher than that of the autotrophically grown cells. The fact that gly-cogen accumulates in fructose grown cells, to a simi-lar extent in the light and in the dark, indicates that the cells contain an abundance of endogenous sub-strates for respiration in both cases. The increased respiratory activity of cells grown in the presence of fructose in the dark, might be due to regulatory func-tions of fructose which are expressed specifically in the dark, and may involve increased levels of re-spiratory chain components.

The fructose-supported DCMU-insensitive cell growth should involve high rates of fructose uptake, not only in the dark but also in the light. Direct measurements of the uptake of [14C]fructose by auto-trophically grown cells of A. azollae were carried out for 15 min periods. Table II shows that cells grown in the absence of fructose, exhibited high rates of fructose uptake, compared to previously reported measurements in other cyanobacteria [6]. Fructose uptake in the light was only partially inhibited by DCMU. Rates of uptake in the dark were also high, amounting to 60% of those measured in the light, indicating that respiration as well as photosynthesis

Table II. Fructose uptake by Anabaena azollae.

Conditions Fructose uptake [(imol g dry w t _ I -h r - 1 ]

light 443 ± 33 light + 10 (ÍM DCMU 297 ± 49 light + 40 |IM CCCP 93 ± 45 dark 258 ± 75 dark + 40 UM CCCP 5 ± 3

Cells were grown autotrophically for four days in BG-11 medium. Fructose uptake was analyzed as described in Materials and Methods.

can supply the energy requirement for fructose up-take. CCCP, which is a potent uncoupler of both oxidative and photosynthetic phosphorylation, inhib-ited 80 percent of the activity in the light and inhibit-ed the activity in the dark almost completely, at a concentration of 40 pM. These results thus demon-strate the operation of a constitutive mechanism for fructose uptake in A. azollae cells. Fructose uptake in cells grown for four days in the presence of fruc-tose was two fold higher than in the control, auto-trophically grown cells. This may indicate further in-duction or activation of the uptake mechanism by its substrate. The incorporation of glucose and sucrose was also tested: the rate of glucose uptake was half of that of fructose, while sucrose uptake rate was only 15 percent of that of fructose.

The results presented in this report demonstrate the capacity of A. azollae cells to incorporate and metabolize fructose for growth both in the dark and in the light. The cells possess an efficient uptake mechanism for fructose, supported by energy provided by either photosynthesis or respiration. Further enhancement of uptake activity could be induced by the presence of fructose in the growth medium. Fructose supported growth was accom-panied by enhanced respiration, decreased 0 2 evolu-tion in the light but increased PS I activity. The bal-ance of these changes should be an overall increase of the potential for ATP synthesis both in the light and in the dark.

The changes induced by the introduction of fruc-tose into the growth medium of A. azollae may be analogous to the sequence of events triggered by the host photosynthates in the cyanobiont in the leaf cav-ity. The changes in respiratory and photosynthetic activities described above should result in reduced

412 A. Rozen et al. • Fructose-Supported Development of Anabaena azollae 412

d e p e n d e n c e on se l f -produced pho tosyn tha t e s , and also in r educed oxygen concent ra t ions . A reduc t ion in pho tosyn the t i c C 0 2 f ixat ion was indeed r e p o r t e d f o r f reshly isolated A. azollae [19]. G lycogen ac-cumula t ion at an early phase of growth in the pres-ence of f ruc tose should result in an increase in the C / N ra t io and suppor t he terocysts d i f fe ren t i a t ion .

Th i s , c o m b i n e d with the decrease in 0 2 concen t ra -t ion , p rov ides the condi t ions necessary to s t imula te n i t rogenase express ion and activity.

Acknowledgement

W e t h a n k Prof . D r . P. B ö g e r fo r critically read ing t h e manusc r ip t .

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